Proliferation of hepatocyte precursors

ABSTRACT

A composition which comprises an animal cell population which contains immature animal cells. The immature animal cells are characterized by expression of alpha-fetoprotein or lack of essential expression of alpha-fetoprotein and albumin, and at least a portion of said immature animal cells or at least a portion of the progeny of said immature animal cells is capable of differentiating into cells which express albumin. The cell population is cultured under conditions which result in expansion of the cells. Expansion of the cells may be achieved by culturing the cells in the presence of an extracellular matrix and liver stromal cells; and preferably in the presence of growth factors. Such cells may be used for liver transplantation, artificial livers, and for toxicology and pharmacology studies. Such cells may also be genetically engineered to express proteins or polypepetides of interest.

This application is a Continuation of U.S. application Ser. No.09/534,487, filed Mar. 24, 2000, incorporated herein by reference in itsentirety, which is a Continuation of U.S. application Ser. No.09/115,920, filed Jul. 15, 1998, incorporated herein by reference in itsentirety, which is a Continuation of U.S. application Ser. No.08/751,546, filed Nov. 18, 1996, incorporated herein by reference in itsentirety, which is a Divisional of U.S. application Ser. No. 08/265,696,filed Jun. 24, 1996, incorporated herein by reference in its entirety,which is a Continuation of U.S. application 07/741,128, filed Aug. 7,1991, incorporated herein by reference in its entirety.

This invention relates to the expansion, or proliferation of cells andin particular cells whose progeny may differentiate into maturehepatocytes. More particularly, this invention relates to the enrichmentof, and to the expansion or proliferation of such cells in the presenceof stromal cells, an extracellular matrix, and/or growth factors. Inanother aspect, this invention relates to genetically engineered cellswhich are capable of differentiating into hepatocytes.

In accordance with an aspect of the present invention, there is provideda composition which comprises an animal cell population. The cellpopulation contains immature cells (i) at least a portion of said cellsor a portion of the progeny of said cells is capable of differentiatinginto hepatocytes and (ii) which are characterized by expression ofalpha-fetoprotein or lack of essential expression of alpha-fetoproteinand albumin, and at least a portion of said cells or of the progeny ofsaid cells is capable of differentating into cells which expressalbumin. In general, the differentiated cells which express albumin havemorphological and physiological characteristics of mature hepatocytes.The cell population has been cultured under conditions which result inexpansion of the immature cells. Such cells are sometimes hereinafterreferred to as “hepatocyte precursors”.

The hepatocyte precursors may be derived from any animal, preferablyfrom mammals. Mammals from which the hepatocyte precursors may bederived include, but are not limited to, humans, rodents (e.g., rats,mice, hamsters), rabbits, bovines, horses, pigs, and sheep. Preferably,the hepatocyte precursors are derived from humans.

Although the hepatocyte precursors are preferably obtained from livertissue, such cells may be obtained from other sources, such as, but notlimited to, the pancreas, gut, lung, and bone marrow.

In general, such hepatocyte precursors may be obtained from an excisedsection of liver. The excised section of liver may then be dissociatedby standard procedures into single dissociated cells. Such proceduresinclude enzymatic dissociation and mechanical dissociation. Enzymaticdissociation may be carried out in the presence of protease(s), such ascollagenase(s) and/or nuclease(s), such as DNase. In some instances,pronase(s) may also be used. Such pronase(s) also contribute to theenrichment of hepatocyte precursors. An example of enzymaticdissociation of liver cells is described in Pretlow, et al., eds., CellSeparation: Methods and Selected Applications, pgs. 45-77, AcademicPress, New York (1987). The cells are then subjected to an enrichmentprocedure to eliminate mature liver cells from the cell population.Various procedures exist for enrichment. Such procedures include, butare not limited to, enzymatic digestion with pronase, DNase, andcollagenase; centrifugal elutriation for cells which are smaller thanmature hepatocytes; and freezing the cells in liquid nitrogen in thepresence of 10% glycerol. It is to be understood, however, that thescope of the present invention is not to be limited to cells of aspecific size range or a specific morphology.

Alternatively, the immature cells may be enriched by contacting cellsfrom an excised section of liver tissue, or of other tissue, which maycontain the hepatocyte precursor cells, with monoclonal antibodies whichrecognize an epitope of the hepatocyte precursor cells. Such cells maythen be separated from the remainder of the cells of the excised tissueby procedures known to those skilled in the art.

One example of an enrichment procedure entails obtaining a liversection, and placing the liver section in an ice-cold saline solutionwhich may contain buffers, glucose, and/or antibiotics. The liversection is then minced and sequentially digested with a solutioncontaining collagenase, pronase, and deoxyribonuclease, prepared in asaline solution to which CaCl 2 is added. The digestions preferably aredone at 37° C. in a shaking water bath and for a period of time of about20 minutes. The partially digested tissue is then strained through atissue sieve by gravity and the undigested remnants are redigested twotimes as hereinabove described. The collected cells are then washed withsaline solution, counted, and assessed for viability.

The enriched hepatocyte precursor population may then be cultured underconditions which result in the expansion, or proliferation of thehepatocyte precursors. Thus, in accordance with another aspect of thepresent invention, there is provided a process for expanding, orproliferating immature cells characterized as hereinabove described. Theprocess comprises culturing the immature cells under conditionsproviding for expansion of the immature cells. Preferably, the processcomprises culturing the immature cells in the presence of (i) anextracellular matrix and (ii) liver stromal cells. Preferably, the liverstromal cells are embryonic liver stromal cells or fetal liver stromalcells. In general, stromal cells are mesenchymally-derived cells that invivo are closely associated with and are in a paracrine relationshipwith epithelia. Stromal cells also grow readily in culture on tissueculture plastic and in serum-supplemented media. In general, such cellsalso produce fibrillar collagens.

The term “expanding”, as used herein, means that the immature cells, orhepatocyte precursors, are cultured under conditions which result in thegrowth or proliferation of the immature cells.

Examples of extracellular matrix components include, but are not limitedto collagen, such as, for example, collagen Type IV, or the adhesionproteins fibronectin, and laminin. A preferred extracellular matrixcomponent is collagen Type IV. The collagen, when employed, may be usedalone or in combination with laminin or fibronectin, or in combinationwith proteoglycans, or with tissue extracts enriched in extracellularmatrix materials.

Preferably, the extracellular matrix component is coated upon a poroussolid support. Examples of porous solid supports which may be employedinclude, but are not limited to porous supports such as Millicellmembrane supports, filters, sponges, and hollow fiber systems.Alternatively, the extracellular matrix may be unattached to the poroussolid support. Examples of such matrices include floating collagen gels,gel foams, spheres of synthetic materials or—fibers of syntheticmaterials such as dextran, polystyrene, and agarose.

The hepatocyte precursors are cultured in a suitable basal medium,preferably a serum-free medium. More preferably, the medium has acalcium content of less than 0.4 mM. Examples of such media include, butare not limited to, Ham's F10, Ham's F12, and RPMI 1640. In a preferredembodiment, the basal medium may further include at least one growthfactor. Growth factors which may be employed include, but are notlimited to, interleukins, such as interleukin-1 and interleukin-3;fibroblast growth factors; prolactin; growth hormone; transforminggrowth factors such as transforming growth factor-∝; insulin-like growthfactors, such as IGF-I and IGF-II; glucagon; insulin; platelet-derivedgrowth factor; thyroid hormones, such as T3; hepatopoietins such ashepatopoietin A and hepatopoietin B; epidermal growth factors (EGF);dexamethasone; norepinephrine; and transferrin. One or more of suchgrowth factors may be contained in a serum-free medium referred to ashormonally-defined medium, or HDM. An example of HDM is furtherdescribed in Enat, et al., Proc. Nat. Acad. Sci., Vol. 81, pgs.1411-1415 (1984). Representative examples of hormonally defined medium(HDM), which may be prepared in RPMI 1640, Ham's F1O, Ham's F12, orother basal media, include the following components in the followingconcentrations:

Component HDM Concentration Insulin 10 μg/ml Growth hormone 10 μU/mlProlactin 20 mU/ml Glucagon 10 μg/ml EGF 50 ng/ml Dexamethasone 10⁻⁸M T310⁻⁹M Selenium 3 × 10⁻¹⁰M Copper 10⁻⁷M Zinc 10⁻¹⁰M

The basal medium may further include a supplement such as, for example,bovine serum albumin, lipoproteins such as high density lipoproteins(HDL), and/or free fatty acids. Free fatty acids which may be containedin the supplement include, but are not limited to, palmitic acid,palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenicacid, or mixtures thereof.

An example of a medium which contains such supplements contains Ham'sF12 medium to which is added 0.4% bovine serum albumin, and 7.6 mEq perliter of a free fatty acid mixture having the following free fatty acidsin the following proportions:

Palmitic acid   31% Palmitoleic acid  2.8% Stearic acid 11.6% Oleic acid13.4% Linoleic acid 35.6% Linolenic acid  5.6%Such medium is sometimes hereinafter referred to as medium HBF.

As a further representative example, either Ham's F12 medium or mediumHBF may include the following growth factors, hormones, or otherchemicals used as supplements in the concentrations given below:

Component Concentration Dexamethasone 10⁻⁶M Insulin 0.1 to 100 μg/mlMulti-Stimulating activity (MSA)* 50 ng/ml EGF 25 to 100 ng/mlNorepinephrine 10⁻⁴M Hepatopoietins 25 μl/ml FGF's 10 ng/ml *Obtainedfrom Sigma Chemical Co., St. Louis, Mo. MSA contains insulin-like growthfactor-I and insulin-like growth factor-II.

The number of such immature cells cultured under conditions such asthose hereinabove described may be monitored by a variety of procedures.In general, the number of such cultured immature cells can increase byat least about 3-fold in a period of one week, preferably at least about10-fold.

In one preferred embodiment, a human liver cell population, which hasbeen enriched for hepatocyte precursors, is plated on or in a matrix ofcollagen Type IV under conditions in which the cells could polarize andfeed through a basal surface such as a Millicell support. Thematrix-bound hepatocyte precursors would be provided with an embryonicliver-derived stromal cell feeder layer. The cells are cultured in aserum-free medium having less than 0.4 mM calcium, and rich in freefatty acids. Some expansion of the hepatocyte precursors occurs undersuch conditions. If one desires to accelerate the expansion of thehepatocyte precursors, one may add growth factors to the medium.Extensive growth, or expansion may be obtained by adding cytokines suchas interleukin-3 or interleukin-1 to the medium. The addition of growthfactors such as, for example, epithelial growth factor (EGF), fibroblastgrowth factor (FGF), or IGF-II induces the expansion of a higherproportion of hepatocyte precursors.

Although Applicants have disclosed herein examples of preferredembodiments for the expansion of the immature cells, it is alsocontemplated that within the scope of the present invention, othermethods may be employed for the expansion of such cells.

Applicants have found that by growing hepatocyte precursors in a mediumwhich contains liver stromal cells and an extracellular matrix, one isable to support, or expand or proliferate the hepatocyte precursors. Onemay obtain growth en masse of the cells (i.e., diffuse, proliferativegrowth), or in some cases, the growth of colonies of such immaturecells, also known as clonal growth, as well as enabling the cells tosurvive for extended periods of time.

Upon expansion of the hepatocyte precursors, such expanded hepatocyteprecursors may be cultured under conditions which enable at least aportion of the hepatocyte precursors or at least a portion of theprogeny of such hepatocyte precursors to differentiate into maturehepatocytes; alternatively, such expanded hepatocyte precursors may betransplanted into a patient, preferably within the liver tissue,whereupon at least a portion of the hepatocyte precursors or a portionof the progeny of such hepatocyte precursors will differentiate intomature hepatocytes.

It is also contemplated that within the scope of the present invention,such hepatocyte precursors may be genetically engineered to express anyof a wide variety of proteins or polypeptides.

Gene(s) of interest which may be expressed by the hepatocyte precursors,or the differentiated cells derived therefrom, include, but are notlimited to: (1) gene(s) present in and expressed at biologicallyeffective levels by normal liver cells, but present in and expressed inless than normal quantities in the liver cells of animals or humanpatients to be treated prior to transfer of gene(s) of interest intothem; (2) gene(s) not expressed in normal mature liver cells; or (3)gene(s) expressed in normal mature liver cells but whose structure isdefective in the animals or patients to be treated, leading to theproduction of a non-functional protein, alone or in any combinationthereof.

The gene(s) of interest can be incorporated into the cellular geneticmaterial (e.g., into genomic DNA) or can be present extrachromosomally(i.e., the gene persists as part of an episome and is expressed from theepisome). The genetic material of interest can be DNA or RNA; the DNAcan constitute all or a portion of a gene of interest (i.e., one whoseexpression in mature liver cells is desired).

The gene(s) incorporated into and expressed by the hepatocyte precursorsor the differentiated cells derived therefrom can additionally includegenetic material (e.g., DNA) encoding a selectable marker, whichprovides a means by which cells expressing the gene(s) of interest canbe identified and selected. Hepatocyte precursors containingincorporated genetic material (i.e., gene(s) of interest and,optionally, genetic material encoding a selectable marker) are referredto as transduced hepatocyte precursors or genetically engineeredhepatocyte precursors.

The gene(s) can be introduced, by means of an appropriate vector, intoisolated and/or cultured hepatocyte precursors, which are subsequentlytransplanted into the recipient. Alternatively, a vector whichrecognizes a hepatocyte precursor as a target may be injected into therecipient, whereby the vector is incorporated into the hepatocyteprecursors.

Such hepatocyte precursors to be genetically modified ex vivo can beobtained from a human or non-human animal, modified and returned to thesame human or non-human animal by transplanting or grafting or,alternatively, can be obtained from a donor (i.e., a source other thanthe ultimate recipient), modified and placed into a recipient, again bytransplanting or grafting.

The genetically engineered cells of the present invention may beemployed in treating any disease which results from a single gene defectwhich can be corrected by expression of the normal gene in thehepatocyte precursors or the differentiated cells derived therefrom.Genetically engineered cells of the present invention may be used, forexample, for the delivery of polypeptides or proteins which are usefulin prevention and therapy of an acquired or an inherited defect inhepatocyte (liver) function. For example, they can be used to correct aninherited deficiency of the low density lipoprotein (LDL) receptor,and/or to correct an inherited deficiency of ornithine transcarbamylase(OTC), which results in congenital hyperammonemia.

Such genetically engineered cells may also be employed in the treatmentof hemophilia due to Factor VIII or Factor IX deficiency; alpha-1anti-trypsin deficiency; phenylketonuria (PKU) or other illnessesresulting from defects in the urea cycle or other defects in metabolicpathways.

The genetically engineered hepatocyte precursors can be used to providea desired therapeutic protein or peptide by a means essentially the sameas that by which the protein or peptide is normally produced and, in thecase of autologous grafting, with little risk of an immune response andgraft rejection. In addition, there is no need for extensive (and oftencostly) purification of a polypeptide before it is administered to anindividual, as is generally necessary with an isolated polypeptide. Suchgenetically engineered hepatocyte precursors produce the polypeptide asit would normally be produced.

Retroviral vectors may be used to transduce hepatocyte precursors withgenetic material which includes gene(s) encoding polypeptide(s) orprotein(s) of interest and/or genetic material encoding a dominantselectable marker.

Because genes can be introduced into hepatocyte precursors using aRetroviral vector, they can be under (subject to) the retroviral vectorcontrol; in such a case, the gene of interest is transcribed from aretroviral promoter. A promoter is a specific nucleotide sequencerecognized by RNA polymerase molecules that start RNA synthesis.Alternatively, retroviral vectors having additional promoter andregulatory elements (in addition to the promoter which is responsiblefor normal retroviral gene transcription), which are responsible for thetranscription of the gene(s) of interest, can be used. This categoryincludes, but is not limited to, promoters, enhancers, or otherregulatory elements for genes normally expressed in the liver. Forexample, a construct in which there is an additional promoter modulatedby an external factor or signal can be used, making it possible tocontrol the level of polypeptide being produced by the modifiedhepatocyte precursors, or by mature hepatocytes which havedifferentiated from such precursors, by providing that external factoror signal. For example, heat shock proteins are proteins encoded bygenes in which the promoter is regulated by temperature. In anotherexample, the promoter of the gene which encodes the metal-containingprotein metallothionine is responsive to cadmium (Cd) ions. Additionalexamples include promoters known to be responsive to cyclic AMP, or toglucocorticoids, or to interferons. Incorporation of these promoters orother promoters influenced by external signals also makes it possible toregulate the production of the polypeptide by the genetically engineeredhepatocyte precursors or mature hepatocytes differentiated from suchprecursors.

It is also possible to use viruses other than retroviruses togenetically engineer or modify hepatocyte precursors. Gene(s) ofinterest can be introduced by means of any virus, or a vector derivativethereof which can express such gene(s) in such cells. For example, SV40,herpes virus, adenovirus, adeno-associated virus, Epstein-Barr virus,and papilloma virus can be used for this purpose. DNA viruses or theirvector derivatives can also be used to introduce gene(s) of interest, aswell as a gene encoding a selectable marker, into such immature cells.

It is also contemplated that the hepatocyte precursors may be transducedwith non-viral expression vehicles or DNA constructs, such as plasmids,for example.

Hepatocyte precursors expressing the gene(s) of interest may be grown intissue culture vessels; removed from the culture vessel; and introducedinto the body. This can be done surgically, for example. In this case,the tissue which is made up of transduced hepatocyte precursors capableof expressing the nucleotide sequence of interest is grafted ortransplanted into the body. For example, it can be placed in theabdominal cavity in contact with/grafted onto the liver or in closeproximity to the liver. Alternatively, the genetically engineeredhepatocyte precursors can be attached to a support, such as, forexample, microcarrier beads, which are introduced (e.g., by injection)into the peritoneal space of the recipient. Direct injection ofgenetically engineered hepatocyte precursors into the liver or othersites is also contemplated. Alternatively, the genetically engineeredhepatocyte precursors may be injected into the portal venous system ormay be injected intrasplenically. Subsequent to the injection of suchcells into the spleen, the cells may be transported by the circulatorysystem to the liver. Once in the liver such cells may express thegene(s) of interest and/or differentiate into mature hepatocytes whichexpress the gene(s) of interest.

Once introduced into the body of an individual, a portion of thegenetically engineered hepatocyte precursors or a portion of theirprogeny differentiates into mature hepatocytes, which provide acontinuous supply of the protein, polypeptide, hormone, enzyme, or drugencoded by the gene(s) of interest. It is contemplated that suchproteins, polypeptides or hormones may also be supplied by thehepatocyte precursors prior to, or in the absence of differentiationinto mature hepatocytes. The amount of the protein, polypeptide,hormone, enzyme, or drug supplied in this way can be modified orregulated as needed.

Thus, the hepatocyte precursors are genetically engineered in such amanner that they produce a gene product (e.g., a polypeptide or aprotein) of interest in biologically significant amounts. The hepatocyteprecursors or the mature hepatocyte progeny therefrom, formed in thisway can serve as a continuous drug delivery system to replace presentregimens, which require periodic administration (by ingestion,injection, etc.) of the needed substance.

Genetically engineered hepatocyte precursors may be employed in thetreatment of inherited disease and in the treatment of acquired disease.In the case of inherited diseases, this approach is used to providegenetically engineered hepatocyte precursors or mature hepatocytesdifferentiated therefrom, which contain DNA encoding a protein orpolypeptide which an individual is unable to make correctly. Hepatocyteprecursors of the present invention can also be used in the treatment ofgenetic diseases in which a product (e.g., LDL receptor) normallyproduced by the liver is not produced or is made in insufficientquantities. Here, hepatocyte precursors transduced with a DNA encodingthe missing or inadequately produced substance can be used to produce itin sufficient quantities. In this case, at least a portion of thetransduced hepatocyte precursors or a portion of their progenydifferentiates into mature hepatocytes, which would produce LDLreceptors and thus provide a means of preventing or treating familialhypercholesterolemia. This is an inherited disease in which the primarygenetic defect is an abnormality in the expression or function of thereceptor for low density lipoproteins, leading to elevated levels ofserum cholesterol and the premature development of coronary arterydisease. The transduced hepatocyte precursors, and the maturehepatocytes differentiated therefrom could be used to produce sufficientquantities of the LDL receptor to overcome the underlying defect. Thisapproach may also be extended to any patient having a predisposition toatherosclerosis due to hyperlipidemia.

There are also acquired diseases for which treatment can be providedthrough use of genetically engineered hepatocyte precursors. Thegenetically engineered hepatocyte precursors may also be employed totreat viral hepatitis, particularly hepatitis B or nonA-nonB hepatitis,by gene transfer. For example, a gene encoding an anti-sense gene couldbe introduced into hepatocyte precursors to inhibit viral replication.In this case, a vector including a structural hepatitis gene in thereverse or opposite orientation would be introduced into hepatocyteprecursors, resulting in production in the genetically engineeredhepatocyte precursors and any mature hepatocytes differentiatedtherefrom of an anti-sense gene capable of inactivating the hepatitisvirus or its RNA transcripts. Alternatively, the hepatocyte precursorsmay be transduced with a gene which encodes a protein, such as, forexample, ∝-interferon, which may confer resistance to the hepatitisvirus.

Advantages of employing hepatocyte precursors of the present inventioninclude the provision of a model system for the growth of hepatocyteprecursors and/or the differentiation of such hepatocyte precursors intomature hepatocytes. Such a model system of hepatocyte precursors hasgreater growth potential than cultures of mature hepatocytes, and thusis better suited for various studies of liver cells, such as toxicologystudies, carcinogenic studies, and vaccine production. Also, becausesuch hepatocyte precursors may be dissociated from liver tissue and thenbe enriched and expanded, such expanded hepatocyte precursors obtainedfrom one liver may thus be administered therapeutically to a pluralityof patients. The administration of such immature cells may also be lesslikely to stimulate immune rejection than the injection of maturehepatocytes. In addition, mature hepatocytes may have a limited lifespan and may undergo a limited number of cell divisions, whereashepatocyte precursors have a greater capacity to generate daughtercells. Thus, the life span of such a system may be significantlyprolonged and possibly may be indefinite.

Examples of non-therapeutic uses of hepatocyte precursors includeresearch of liver embryology, liver cell lineages, and differentiationpathways; gene expression studies; mechanisms involved in liver injuryand repair; research of inflammatory and infectious diseases of theliver; studies of pathogenetic mechanisms; and studies of mechanisms ofliver cell transformation and etiology of liver cancer. Additionaltherapeutic uses include liver transplantation for patients with liverfailure due to alcoholism, infection, congenital liver diseases, etc.,gene therapy for liver diseases that are genetically based such as, forexample, Wilson's disease, glycogen storage diseases, urea cycle enzymedefects, and Creigler-Najir disease; and the use of such hepatocyteprecursors and any lineages of adult cells derived from them in assaysfor chemotherapy (eg., for liver cancers), for the production ofvaccines for viruses that grow in the liver, and for studies ofalcoholic cirrhosis. The hepatocyte precursors cells may also beemployed as part of an “artificial liver;” i.e., the hepatocyteprecursors may be placed in a container or apparatus, in which thehepatocyte precursors generate a liver lineage and function as a liveroutside of the body. The container or apparatus is connected to thecirculatory system of a human or animal subject.

In accordance with another aspect of the present invention, there isprovided a composition comprising an animal cell population derived fromliver. The cell population contains immature cells which arecharacterized by expression of alpha-fetoprotein or lack of essentialexpression of alpha-fetoprotein and albumin, and at least a portion ofsuch cells or of the progeny of such cells is capable of differentiatinginto adult liver cells. The cells have been cultured under conditionswhich result in expansion of the immature cells. Such immature cells maybe obtained from the livers of human or non-human animals hereinabovedescribed. Although the progeny of such immature cells may differentiateinto hepatocytes, such immature cells may differentiate into adult livercells other than hepatocytes, such as bile duct cells, liver endothelialcells, and lipid-containing liver cells known as Ito cells.

Such immature cells derived from liver may be obtained from liver tissueand enriched or expanded under conditions hereinabove described for theenrichment and expansion of the above-described hepatocyte precursors.It is also contemplated that such immature cells may be geneticallyengineered through techniques such as those hereinabove described,whereby such genetically engineered cells may be administered to ananimal or a human subject, in which the genetically engineered cellsand/or their differentiated progeny express gene(s) of interest.

It is to be understood, however, that the scope of the present inventionis not to be limited to the specific embodiments described above. Theinvention may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

1. A composition comprising a cell culture of isolated immature livercells, which contains at least a population of hepatocyte precursorcells capable of differentiating into hepatocytes.
 2. The composition ofclaim 1, wherein the hepatocyte precursor cells are capable ofdifferentiating into hepatocytes in a serum-free culture mediumcomprising extracellular matrix and liver stromal cells.
 3. Thecomposition of claim 2, wherein the extracellular matrix is formed froma material comprising collagen, fibronectin, laminin or combinationsthereof.
 4. The composition of claim 3, wherein the collagen is type IVcollagen.
 5. The composition of claim 2, wherein extracellular matrix isformed of collagen optionally in combination with proteoglyeans ortissue extracts enriched in extracellular matrix materials.
 6. Thecomposition of claim 2, wherein the extra cellular matrix is coated upona porous solid support.
 7. The composition of claim 6, wherein the solidsupport comprises Millicell membrane support, filters, sponges, andhollow fiber systems.
 8. The composition of claim 2, wherein the liverstromal cells are embryonic liver stromal cells.
 9. The composition ofclaim 2, wherein the liver stromal cells are fetal liver stromal cells.10. The composition of claim 1 which comprises a growth factor. 11.Genetically engineered hepatocyte precursor cells obtained bygenetically engineering expanded hepatocytes precursor cells derivedfrom culturing isolated immature liver cells that contain at least apopulation of hepatocyte precursor cells capable of differentiating intohepatocytes.
 12. The genetically engineered hepatocyte precursor cellsof claim 11, wherein the hepatocyte precursor cells are differentiatedinto hepatocytes in a serum-free culture medium comprising extracellularmatrix and liver stromal cells.
 13. Genetically engineered hepatocyteprecursor cells obtained by culturing isolated immature animal cellsincluding liver, pancreas, gut, lung, or bone marrow cells, that containat least a population of hepatocyte precursor cells capable ofdifferentiating into hepatocytes in a serum free culture medium, thatcomprises extracellular matrix and liver stromal cells to provideexpanded hepatocyte precursor cells and genetically engineering theexpanded hepatocyte precursor cells.
 14. The genetically engineeredhepatocyte precursor cells of claim 13, wherein the liver stromal cellsare embryonic liver stromal cells or fetal liver stromal cells.
 15. Thegenetically engineered hepatocyte precursor cells of claim 11, whereinthe genetic engineering comprises ex vivo genetic modification of thehepatocyte precursors.
 16. The genetically engineered hepatocyteprecursor cells of claim 11, wherein genetically engineering comprisestransducing hepatocyte precursor cells with a retroviral vectorcomprising a genetic material that encodes polypeptides or protein ofinterest and/or a dominant selectable marker.
 17. The geneticallyengineered hepatocyte precursor cells of claim 11, wherein the geneticmaterial is under the control of retroviral vector regulatory elementsand/or regulatory elements of genes normally expressed in the liver.